PROJECT SUMMARY DNA methylation, histone modification, and the machinery involved in epigenetic regulation are key to maintaining proper cellular homeostasis. A common feature among cancer and other diseases is the misregulation of epigenetic marks in CpG-rich promoter regions. Aberrant hypermethylation of promoter CpG islands and the resulting gene silencing is an established mechanism for the inactivation of tumor suppressor genes. The methylated DNA is recognized by a set of proteins called methyl-CpG-binding domain (MBD) proteins, which recruit modification proteins for chromatin organization and epigenomic maintenance, leading to alterations of the DNA architecture and inappropriate repressive heterochromatin formation. Studies have shown a link between the occupancy of MBD proteins and the hypermethylation status of tumor suppressor genes, which contributes to tumorigenesis. Knockout studies targeting MBD proteins have demonstrated therapeutic targeting potential; however, small molecule inhibitors of MBD proteins currently do not exist. The overall objective of this proposal is to develop small molecule pyrrole-imidazole (Py-Im) polyamides targeted to hypermethylated CpG promoter regions of tumor suppressor genes in human cancer cell lines, to disrupt the MBD protein- DNA binding. By interfering with MBD protein-DNA binding, we aim to regain expression of silenced tumor suppressor genes due to misregulation of epigenetic marks and subsequent chromatin architecture. Previous work has demonstrated the ability of Py-Im polyamides to de-silence gene expression by preventing repressor-DNA interactions. In this work, a library of sequence-specific Py-Im polyamides targeted to CpG-rich DNA sequences will be designed, synthesized and tested for their ability to inhibit MBD protein-DNA binding. Effects of this inhibition in cell cultures will be examined in the context of gene- specific and genome-wide mRNA expression, as well as cell proliferation and viability, to assess the genome-wide specificity of polyamide-mediated gene regulation. This proposal describes a novel, non-mutagenic approach to combat epigenetic-driven cancers. The de-silencing of tumor suppressor genes by inhibition of MBD proteins at the protein-DNA interface represents a new approach to cancer treatment. By examining the activity, mode of action, specificity and outcome of targeting MBD proteins, this study will provide a better understanding of MBD protein-dependent gene silencing in tumorigenesis; we believe these efforts will represent a valuable contribution to the development of gene-targeted systemic treatments for cancer therapies. The direct targeting of DNA-binding, without causing damage to the DNA, represents a powerful and highly specific approach to fighting cancer, and if successful will directly benefit public health.